1. Field of the Invention
The invention in the field of biochemistry and medicine relates to improved chimeric HIV envelope protein (Env) immunogen or vaccine compositions in which an Env-encoding nucleic acid includes three different Env V3 loop sequences.
2. Description of the Background Art
The strains of HIV-1 can be classified into three groups: the “major” group M, the “outlier” group 0 and the “new” group N. These three groups may have resulted from three separate introductions of simian immunodeficiency virus into humans. Group 0 appears to be restricted to west-central Africa and group N—discovered in 1998 in Cameroon—is extremely rare. More than 90% of HIV-1 infections belong to HIV-1 group M. Within group M there are known to be at least nine genetically distinct clades or subtypes of HIV-1: clades A, B, C, D, F, G, H, J and K.
Occasionally, two viruses of different subtypes can meet in the cell of an infected person and mix together their genetic material to create a new hybrid virus. Many of these new strains do not survive for long, but those that infect more than one person are known as “circulating recombinant forms” or CRFs. For example, the CRF A/E is a mixture of genes in one virus from clades A and E.
The classification of HIV strains into lades and CRFs is complex, and the definitions are subject to change as new discoveries are made. Some scientists refer to clades or subtypes A1, A2, A3, F1 and F2 instead of A and F, though others regard the former as sub-clades or sub-subtypes.
Many HIV-1 neutralizing antibodies in infected individuals or in immunized animals are directed against the V3 loop of the viral envelope protein gp120 which was accordingly designated the principal neutralizing determinant of HIV-1 (Rusche, J R et al (1988) Proc. Natl. Acad. Sci. USA 85: 3198-3202). HIV-neutralizing antibodies against V3 are thought to prevent the binding of gp120 to either R5 or X4 co-receptors, thus abolishing fusion of the virus with its target cell.
The present inventors and colleagues developed the human mAb 447-52D (IgG3, λ) from a heterohybridoma derived from peripheral blood mononuclear cells of a clade B HIV-1-infected individual (Gorny, M K et al. (1993) J. Immunol. 150, 635-643). Monoclonal antibody 447-52D is one of the most broadly neutralizing and most potent anti-V3 antibodies that has been studied to date. It binds to intact virions from clades A, B, C, D, F, G and H (Nyambi, P N et al. (1998) J. Virol. 72, 9384-9391) and neutralizes primary isolates from several clades, including both X4 and R5 type viruses (Cecilia, D et al. (1998) J. Virol. 72.6988-6996; Conley, A J et al. (1994) J. Virol. 68.6994-7000; Fouts, T R et al. (1997) J. Virol. 71:2779-2785; Gorny, M K et al. (2002) J. Virol. 76:9035-9045; Hioe, C E et al. (1997) Int. Immunol. 9:1281-1290; Nyambi et al., supra; Verrier, F et al. (2001) J. Virol. 75:9177-9186). 447-52D recognizes the V3 loop; its core epitope has been mapped with overlapping peptides to the highly conserved motif at the V3 crown GPxR (residues 319-322) (Gorny M K et al. (1992) J. Virol. 66:7538-7542; Gorny et al., 1993, supra). Unlike most anti-V3 antibodies, 447-52D can neutralize both X4 and R5 primary viral isolates correlating with its ability to bind V3 peptides with a wide range of sequence variability (Zolla-Pazner, S et al. (1999) J Virol 73:4042-4051. 447-42D binds to different V3 peptides with association constants ranging between 2×105 and 108 M−1, the highest of which is only one order of magnitude lower than its affinity for the corresponding (intact) gp120 protein (VanCott, T C et al. (1994) J. Immunol. 153:449-459). Since 447-52D was elicited during the course of a natural HIV-1 infection and neutralizes a broad spectrum of HIV-1 isolates, it is believed to recognize a native V3 conformation.
The presence of neutralizing antibodies in patient sera and the development of monoclonal antibodies such as the 447-52D demonstrates the ability of the human immune response to produce protective antibodies. However, the induction of broadly neutralizing anti-HIV-1 antibodies, more particularly broadly neutralizing anti-V3 antibodies, via immunization of animals and humans has largely been a haphazard matter of chance.
Recent attempts to produce broadly reactive neutralizing antibodies are described in several publications. Chakrabarti B K et al, 2005, Vaccine 23:3434-45, compared Env immunogens with substituted V3 regions to combinations of strains from different clades, evaluating the ability of such immunogens to expand the breadth of the neutralizing antibody response. When the V3 region from HIV BaL was substituted for HIV HXB2, an effective neutralizing antibody response against several clade B primary isolates was elicited, but remained restricted to neutralization of most clade B isolates. In an attempt to expand this response further, a linear epitope recognized by the broadly neutralizing 2F5 antibody was inserted into V3. A V3 epitope was identified that bound to Ab 2F5 and elicited a potent 2F5-like antibody response when administered as an immunogen. However such antisera neutralized only a lab-adapted strain and not primary isolates. In contrast, combinations of Envs from clades A, B, and C, elicited neutralizing antibodies to a more diverse group of primary HIV-1 isolates. The authors suggested that combinations of Env immunogens, despite the limited reactivity of the V3 from each component, can be used to expand the breadth of the neutralizing antibody response.
A multi-envelope HIV-1 vaccine cocktail, containing 51 unique envelope proteins has been tested in six macaques, giving rise to significant neutralization of viruses from clades A, B, and D (Zhan, X et al, 2005; Vaccine 23:5306-20. Epub 2005 Jul. 20). This vaccine was administered as successive immunizations with recombinant DNA, recombinant vaccinia virus and recombinant HIV-1 Env proteins. Following vaccination, animals developed diverse anti-Env antibodies with binding and neutralizing activities toward proteins and viruses that were not represented by sequences in the vaccine. Vaccinated and control animals were challenged with the heterologous pathogen SHIV, 89.6P. The vaccinated monkeys experienced significantly lower virus titers and better maintenance of CD4+ T-cells than did unvaccinated controls. Four of six vaccinated animals but only one of six control animals, survived 44-weeks post-challenge. The authors stated that this was the first report describing control of pathogenic SHIV disease by a heterologous HIV-1 vaccine (devoid of 89.6 or simian immunodeficiency virus derivatives).
Seaman M S, et al., 2005, J Virol. 79:2956-63, examined the magnitude and breadth of envelope (Env)-specific T-lymphocyte and antibody responses generated by vaccines containing either a single or multiple genetically distant HIV-1 Env immunogens. Rhesus monkeys were immunized with DNA prime-recombinant adenovirus boost vaccines encoding a Gag-Pol-Nef polyprotein in combination with either a single Env or a mixture of clade-A, clade-B, and clade-C Envs. Monkeys receiving the multiclade Env immunization developed robust immune responses to all vaccine antigens and, importantly, a greater breadth of Env recognition than monkeys immunized with vaccines including a single Env immunogen. All groups of vaccinated monkeys were infected following challenge with the pathogenic simian-human immunodeficiency virus 89.6P and demonstrated equivalent immune protection in terms of control of viremia. The authors suggested that a multicomponent vaccine encoding Env proteins from multiple clades of HIV-1 can generate broad Env-specific T-lymphocyte and antibody responses without antigenic interference and that it is possible to generate protective immune responses by vaccination with genetically diverse isolates of HIV-1.
In a recent publication by two of the present inventors (Lu and Wang) and their colleagues (Virology, e-published Apr. 6, 2006)), they compared polyvalent vs. monovalent immunization of rabbits. Polyvalent constructs gave better results. Rabbits were first immunized with a DNA vaccine expressing 1, 3 or 8 primary HIV-1 gp120 antigens delivered by gene gun followed by boosting with recombinant gp120 protein. Sera from rabbits immunized with DNA priming plus protein boosting, but not DNA vaccine alone or the Env protein alone, were capable of neutralizing 7 of 10 viruses in one type of assay and 12 of 14 viruses in a second type of assay. Sera from rabbits immunized with the polyvalent Env antigens neutralized a significantly higher percentage of viruses than did sera from rabbits immunized with monovalent antigens. The authors suggested that DNA priming followed by recombinant Env protein boosting can be used to deliver polyvalent Env-antigen-based HIV-1 vaccines to elicit neutralizing antibody responses against viruses with diverse genetic sequence variations.
In the Chakrabarti et al. study, above, the investigators used (i) a “monoclade” immunogen (500 μg of purified plasmid DNA from gp145deltaCFI of HIV-1 HXB2 substituted with the V3 of HTV-1 BAL) or (ii) a multiclade immunogen comprising three different preparations, so that “vaccinees” got 167 μg of each of three plasmids. In the latter case, it is noteworthy that only ⅓ the amount of each V3 epitope was administered, which would be expected to compromise the effort. The present invention differs fundamentally from the approach of Chakrabarti et al., Seaman et al. and Wang et al., supra, in that a single molecule carrying the three different V3 regions is administered, thereby providing to the vaccinee the full complement of each of three V3s.
Other than the few studies cited above, the polyvalent approach using Env antigens from different clades of primary HIV-1 isolates circulating in the world has not been well investigated. A number of difficulties in developing polyvalent Env vaccines are discussed briefly below:
(1) Screening and Selecting Multiple Antigens:
This has been approached in a variety of ways resulting in conflicting conclusions. For example, a report from one of the present inventors (Zolla-Pazner) and colleagues (Nyambi et al., 2000, J. Virol. 74:10670-80) suggested that little or no correlation exists between clade and antigenic characteristics of the HIV-1 gp120 envelope glycoprotein; rather, “immunotypes” were defined which include viruses from diverse clades carrying Env proteins with common antigenic characteristics In contrast, Binley, J M et al., 2004, J Virol. 78:13232-52, suggested that Env antigenic characteristics do correlate with clades. Still others have used an essentially random method of selecting large numbers of virus envelopes in the hopes of covering all or most of the possible virus subtypes. (See, for example Zhan et al., supra).)
(2) Producing Multiple Antigens:
If a polyvalent vaccine is needed, and if each immunogen is represented by a different molecule, the production, quality control and safety issues are multiplied by the number of molecules that will need to be combined into the vaccine.
(3) The Use of Multiple Immunogens May Weaken the Immune Response to Each:
This is exemplified by results of Wang et al., supra, in which a combination of 8 immunogens yielded a weaker immune response than a combination of three. The present inventors' approach disclosed herein is based on the reasoning that an immunogenic molecule that comprises, and can present, multiple copies of relevant antigen(s) would be advantageous as, in essence, such a molecule would introduce several moles of the epitope of interest per mole of immunogen.
(4) None of the Current Approaches Described Above Focus the Immune Response Exclusively to Epitopes that are Known to Induce Neutralizing Antibodies:
Thus, if one immunizes with one or multiple Env molecules as immunogens, the recipient will make antibodies to dozens of epitopes, and the majority of these will not be neutralizing antibodies. Only very few HIV-1 envelope glycoprotein epitopes induce neutralizing antibodies, one of these being V3.